Combined use of carnosinase inhibitor with l-carnosines and composition

ABSTRACT

The present invention provides combined use of a carnosinase inhibitor with L-carnosine and its related substance, and a composition containing the same, which are useful for treatment or prevention of various diseases, improvement of health conditions, improvement of exercise ability, improvement of skin health, prevention of the side effects of alcohol drinking and the like in mammals including human.

TECHNICAL FIELD

This invention relates to combined use of a carnosinase inhibitor withL-carnosine and its related substance, and a composition containing thesame. The combined use of a carnosinase inhibitor with L-carnosine andits related substance, and a composition containing the same are usefulin the filed of pharmaceutical compositions, cosmetics, foods andbeverages.

BACKGROUND ART

Free radicals and the peroxidative processes caused by them are believedto be one of the causes of the structural and functional degradations ofhuman and other mammalian tissues during aging. It is important forprevention or minimization of the aging processes and relatedpathologies, namely for longevity, to maintain the high concentration ofnatural antioxidant molecules (free radical scavengers) inside andaround the body's various tissues and cells, and in the body's liquidsincluding the blood plasma and blood stream in order to supply organs,tissues and cells with the antioxidant molecules.

L-Carnosine (β-alanyl-L-histidine) (I) has been found to be one of themost abundant (1-20 mM) nitrogenous compounds present in the non-proteinfraction of vertebrate skeletal muscles (e.g. non-patent literature 1,2)and certain other tissues, including olfactory epithelium, and bulbs(e.g. non-patent literature 3) and also the crystalline lens (e.g.non-patent literature 4).

Some L-carnosine-related natural substances such asN-acetyl-L-carnosine, L-anserine (β-alanyl-L-1-methylhistidine) and itsacyl derivatives, L-balenine (β-alanyl-L-3-methylhistidine) and itsacetyl derivatives, L-homocarnosine (γ-aminobutyryl-L-histidine) and itsacetyl derivative, and carcinine (β-alanylhistamine) and its acetylderivatives have been also reported (e.g. non-patent literature 5,6,7)to be present at millimolar concentrations in several mammalian tissues,including skeletal muscles, cardiac tissue and brain, although there areinteresting differences in their distribution, activities and metabolictransformation (e.g. non-patent literature 8).

L-Carnosine and its related natural substances mentioned above have beenknown to be among the most important natural antioxidant agents actingboth in the lipid phase of the cellular or biological membranes and inthe aqueous environment, to protect lipids and water-soluble moleculeslike proteins (including enzymes), DNA and other essentialmacromolecules from oxidative damage mediated by reactive oxygen speciesand lipid peroxides (e.g. non-patent literature 9, 10, 11, 12).

Various antioxidant enzymes such as SOD (superoxide dismutase) orcatalase can only react with their substrates in the aqueousenvironment. On the other hand, previously published data suggest thatL-carnosine and its related natural substances have excellent potentialto act as natural antioxidants which scavenge hydroxyl radical,superoxide and singlet oxygen, resulting in protection of lipids fromperoxidation (e.g. non-patent literature 13, 14).

Further detailed explanation on the physiological properties ofL-carnosine and related compounds is as follows:

1) L-Carnosine and its naturally occurring related substances protectthe proprietary cellular and tissue antioxidant enzymes (like SOD) andsystems (like glutathione and ceruloplasmin) from inactivation.L-Carnosine, though water soluble, potentiates the antioxidant affect oflipid soluble alpha tocopherol during lipid peroxidation in the livermicrosomes. It is thus a major protector of the liver cytochrome P-450system. L-Carnosine is also the only known antioxidant to significantlyprotect cellular chromosomes from oxidative damage. These antioxidanteffects are thought to be beneficial to prevent atherosclerosis,ischemic diseases (e.g., brain infarction, transient ischemic arrest,stroke, angina pectoris, cardiac infarction, etc.), formation andprogression of cataracts, and aging of the muscles and the skin.L-Carnosine is also known to ameliorate the neuro-degenerative diseasesof the brain (e.g., Alzheimer's disease, dementia, epilepsy, etc.).Recently it is postulated that a dysfunctioning of the anti-oxidantsystem such as L-carnosine, glutathione and superoxide dismutase (SOD),causes overstimulation of the post-synaptic glutamate receptors (NMDAand AMPA) leading to the expression and progression of schizophrenia,Parkinson's disease, Huntington's chorea, and amyotrophic lateralsclerosis (e.g. non-patent literature 15). It is thought that supplementof L-carnosine is a useful way to prevent and to treat these diseases.

2) L-Carnosine is the most effective anti-glycating agent ever found andis known to prevent the so-called Maillard reaction in both the directglycation of proteins and the advanced glycation such as cross-linkingof the proteins. It is expected, therefore, that L-carnosine preventsnot only diabetic complications such as diabetic retinopathy, diabeticneuropathy, diabetic nephropathy, etc., but also aging of varioustissues such as vascular tissues (atherosclerosis, retinopathy, etc.),skin (wrinkles), lenses (cataracts), and so on. Very recently, it hasalso been reported that a carcinogenic and toxic substance acrylamidewas formed in a variety of foods during cooking through the Maillardreaction (e.g. non-patent literature 16, 17). Addition of L-carnosine topre-cooked foods or foodstuffs is therefore expected as a useful mean toprevent the formation of acrylamide.

3) L-Carnosine has a membrane-stabilizing and tissue-repairing effectsand is effective in protecting and repairing the ulcerative diseases ofthe gastrointestinal tract(e.g. gastric ulcer and duodenal ulcer) andthe skin. It is also effective to heal a wound and a burn.

4) L-Carnosine and its related natural substances have anti-ischemiceffects not only in the brain, but also in the heart. L-Carnosine hasbeen shown to increase the strength of heart contractility by enhancingcalcium response in the heart cells. L-Carnosine injections have beenshown to be effective emergency treatment for ischemic crises.

5) L-Carnosine has been shown to play an important role to control theacid-base balance of the muscles when a large quantity of H⁺ is producedin association with lactic acid accumulation during high-intensityexercise. Thus L-carnosine plays a role to prevent the fatigue of themuscles leading to the improvement of the exercise-performance ability.It protects muscle cell membranes from oxidation under the acidicconditions of muscular exercise. According to a recent report, theL-carnosine concentration in the muscle was significantly correlatedwith the mean power body mass (e.g. non-patent literature 18).L-Carnosine has been shown to dramatically improve exercise recovery(but does not increase performance, which means that it is not an“ergogenic aid, ” but rather facilitates the anabolic response toexercise). Carnosine has been shown to quickly restore musclecontraction capability after fatigue.

6) Recently, it is said that L-carnosine is likely to be the truesubstrate of nitric oxide synthase. The supplement of L-carnosinestimulates the synthesis of nitric oxide (NO) which is an internal bloodvessel relaxing material, and is expected to reduce blood pressure,prevent ischemic diseases like angina pectoris and improve penileerectile dysfunction.

7) L-Carnosine can act as a scavenger of acetaldehyde (non-patentliterature 19) which is the main product of ethanol metabolism upon theconsumption of alcohol-containing drinks (whisky, cognac, vodka, beer,sake, wine, etc.). Thus L-carnosine protects the liver, the brain andmuscles from the toxic effect of the acetaldehyde. L-Carnosine due toits wound healing property is also effective to cure and prevent theulcers in the gastro-intestinal tract which can be exacerbated duringthe alcohol consumption.

8) The chronic form of alcoholic skeletal myopathy is characterized byselective atrophy of Type II fibers and affects up to two thirds of allalcohol misusers. L-Carnosine prevents the atrophy of the fibers.Carnosine protects the brain from both lipid peroxidation and damagecaused by excessive alcohol intake.

9) L-Carnosine has immunostimulating property. It protects the immunesystem from immunosuppression by hydrocortisone, anti-tumor drugs, andmany other immunosuppressive drugs.

10) L-Carnosine has the following benefits for keeping health and youth:L-Carnosine and related compounds increase the number of the celldivisions (Hayflick limit) and may be useful for elongation of the lifespan and for longevity (e,g, non-patent literature 20). L-Carnosine hasa rejuvenating effect on connective tissue cells and a wound healingeffect. L-Carnosine has been shown to rejuvenate cells approachingsenescence by extending the life of those cells which will continue todivide with the frequency typical of youth. In tissue culturessupplemented with L-carnosine, cells retain a youthful appearance andhave an extended cellular life span. This ability for L-carnosine toincrease cellular life span holds true even for old cells. One studyshowed a 67% increase in cellular life span with L-carnosinesupplementation. This property has been proved in vivo by using mice.The mice supplemented with L-carnosine lived an average of 20% longerthan non-treated mice, and kept a healthy state twice more thannon-treated mice when they reached an old age. In humans, L-carnosinelevels decline with age. Muscle L-carnosine concentration decreases 63%during age 10 to age 70.

On the other hand, however, exogenous carnosine, even when topicallyadministered to the eye, does not accumulate in the tissues, because itis easily excreted in urine or destroyed by the enzyme calledcarnosinase, which is present in blood plasma, aqueous humor of theanterior eye chamber, liver, kidney and other tissues, but not in themuscles (e.g. non-patent literature 21, 22)) and probably in the lens(e.g. non-patent literature 23, 24. L-Carnosine-related naturalsubstances such as N-acetyl-L-carnosine, L-anserine,N-acetyl-L-anserine, L-balenine, L-homocarnosine,N-acetyl-L-homocarnosine and carcinine can be more or less thesubstrates of carnosinase to be hydrolyzed and deactivated, although therates of the hydrolysis are slower than that of L-carnosine ( e.g.non-patent literature 25).

As mentioned above, it is predicted that a shortage of L-carnosineand/or its related natural substances in the body as a result of agingor malnutrition, for instance, causes a variety of dysfunction of thebody of humans and other mammals. The supplement of L-carnosine or itsnatural related substances is therefore thought to be useful to treat orprevent these dysfunction or the resulting diseases. It is also expectedthat an excessive supplement of L-carnosine and/or its related naturalsubstances becomes meaningful to cure these diseases, to improveexercise performance, to prevent aging, to care the skin, to improve thequality of life (QOL), and so on. Although L-carnosine is already usedas a dietary supplement or a cosmetic in some countries, these are notthought to be very effective, because, as mentioned above, L-carnosineis easily hydrolyzed to β-alanine and L-histidine by an enzyme calledcarnosinase (in its serum and tissue forms), resulting in the completeloss of the physiological effects of L-carnosine. Since carnosinase iswidely distributed in the lumen of the small intestine, in the plasma,and in the various tissues of humans and mammals, the administration ofa large excess of L-carnosine regardless of the route of the treatment,i.e., oral, parenteral, or topical, has been recommended in order toattain the above-mentioned purposes, namely treatment of diseases,improvement of exercise performance, prevention of aging, skin care,improvement of QOL, and so on, by saturating the enzyme to prevent thecomplete hydrolysis of the L-carnosine administered. However, such alarge excess treatment should be avoided not only from the practical andeconomical point of view, but also from the viewpoint of safety, inparticular due to the risk of histamine release in tissues and bloodplasma. When a large excess amount of L-carnosine is applied, excessivehistamine may be formed in the body by the action of another enzymehistidine decarboxylase(s) on the excessive L-histidine released bycarnosinase. Therefore, development of an alternative and safe way thatmakes the efficient delivery of L-carnosine or its related naturalsubstance(s) to the target tissues possible without its degradation haslong been waited.

On the other hand, there has been no literature concerning the use ofcarnosinase inhibitors to increase the delivery of L-carnosine or itsrelated natural substances to the plasma or the target tissues or organsby preventing it from hydrolysis and deactivation. It is known thatbestatin is a potent tissue carnosinase inhibitor (e.g. non-patentliterature 26), and that accumulation of internal tissue L-carnosine isincreased upon treatment with bestatin. However, it has not been knownif bestatin can effectively help deliver the L-carnosine to the targettissues when L-carnosine and bestatin are administered simultaneously invivo.

[Non-patent literature 1] K. G. Crush, Comp. Biochem. Physiol., Vol. 34,3, 1970;

[Non-patent literature 2] A. A. Boldyrev, S. E. Severin, Adv. EnzymeRegul., Vol. 30, 175, 1990

[Non-patent literature 3] F. Margolis, Science, Vol. 184, 909, 1974

[Non-patent literature 4] Jay et al., Meeting Abstr. J. Physiol. London,Vol. 420, 155, 1990

[Non-patent literature 5] P. R. Carnegie et al., J. Chromatogr., Vol.261, 153, 1983

[Non-patent literature 6] L. Flancbaum et al., Life Sci., Vol. 47, 1587,1990

[Non-patent literature 7] M. A. Babizhayev et al., Biochemistry(Moscow), Vol. 63, 620, 1998

[Non-patent literature 8] M. A. Babizhayev et al., Biochem. J., Vol.304, 509, 1994

-   [Non-patent literature 9] M. A. Babizhayev et al., Biochem. J., Vol.    304, 509, 1994 and references thereof.    [Non-patent literature 10] M. A. Babizhayev et al., Biochemistry    (Moscow), Vol. 63, 523, 1998-   [Non-patent literature 11] M. A. Babizhayev et al., Clinica Chimica    Acta, Vol. 254, 1, 1996-   [Non-patent literature 12] J. H. Kang et al., Molecules and Cells,    Vol. 13, 107, 2002 and references thereof.-   [Non-patent literature 13] T. A. Dahl, W. R. Midden, P. E. Hartman,    Photochem. Photobiol., Vol. 47, 357, 1988    [Non-patent literature 14] M. A. Babizhayev et al., Biochem. J.,    Vol. 304, 509, 1994 and references thereof    [Non-patent literature 15] Encephale, Vol. 28(2), 147, 2002    [Non-patent literature 16] D. S. Mottram, et al, Nature, Vol. 419,    448, 2002    [Non-patent literature 17] R. H. Stadler, et al., Nature, Vol. 419,    449, 2002    [Non-patent literature 18] Y. Suzuki et al., Japan. J of Physiol.,    Vol. 64, 199, 2002    [Non-patent literature 19] Ann. NY Acad. Sci., Vol. 854, 37, 1998-   [Non-patent literature 20] R. Holliday, G. A. McFarland.,    Biochemistry (Moscow), Vol. 65, 843, 2000    [Non-patent literature 21] Jackson et al., Clin. Chim. Acta, Vol.    196, 193, 1991 [Non-patent literature 22] Lenney et al., Biochem.    J., Vol. 228, 653, 1985    [Non-patent literature 23] A. A. Boldyrev, A. Y. Dupin, M. A.    Babizhayev, S. E. Severin, Biochem. Int., Vol. 15, 1105, 1987    [Non-patent literature 24] J. L. Jay et al., Meeting Abstr. J.    Physiol. Lond., Vol. 420, 155, 1990    [Non-patent literature 25] A. Pegova et al., Comp. Biochem.    Physiol. B. Biochem. Mol. Biol., Vol. 127(4), 443, 2000    [Non-patent literature 26] S. C. Peppers, J. F. Lenney, Biol. Chem.    Hoppe Seyler, Vol. 369, 1281, 1988)

DISCLOSURE OF THE INVENTION

This invention relates to the combined use of a carnosinase inhibitorand L-carnosine or its related substances and to the formulationthereof. This invention is useful for treatment and prevention ofL-carnosine-related diseases, improvement of health conditions,improvement of exercise ability, prevention of aging, improvement ofskin health, and prevention of the side effects of alcohol drinking.

The present inventors completed this invention by finding that it isuseful to prevent or treat carnosine-related diseases of mammals whenL-carnosine and a carnosinase inhibitor are used in combination. Theinventors also found that such combined use and formulation thereof areuseful for improvement of health conditions, improvement of exerciseperformance, prevention of aging, improvement skin health, andprevention of the side effects of alcohol drinking.

According to this invention, L-carnosine or its related substancesadministered orally, parenterally or topically (to the skin, eyes,mucous membrane of mouth or nose, respiratory tract, etc.) is deliveredeffectively to the plasma or the target tissues or organs with highbioavailability, when a carnosinase inhibitor is administeredsimultaneously through a route similar to the one used for L-carnosineor its related substances. Any combination of the compounds and theadministration route for each compound may be possible. As the result,the dosage of L-carnosine or its natural derivative to be applied can bereduced or the physiological effects of L-carnosine can be potentiatedmarkedly. It was also found that in order to increase the absorption ofL-carnosine or its related substances and/or a carnosinase inhibitor,addition of at least one of the additives selected from cellulosederivatives, gelatin and polyvinylpyrrolidone is useful. This inventionrelates to these unique and effective methods.

Furthermore, this invention relates to a novel method for providing anew chemotherapy of cancer characterized by administering bestatin, atissue carnosinase inhibitor, together with L-carnosine. Althoughbestatin itself has been known to have anti-cancer effect, the presentinventors found that the anti-cancer effect was improved by thesimultaneous administration of L-carnosine. This is based on thehitherto unknown, unexpected finding that the increase of L-carnosinelevel in the cancer cells results in the prevention of the division ofthe malignant cancer cells without affecting normal cells. The precisemolecular basis of this anti-cancer activity of L-carnosine according tothe data of the present inventors is related to the ferroxidase activityof L-carnosine in the malignant cancer cells, by which the deprivationof ferrous ion concentration in the malignant cancer cells occursinducing their apoptosis, a prompt death of the malignant cancer cells.

The present inventors have made extensive studies on carnosinaseinhibitors and discovered β-alanine (II), N-alkanoyl-β-alaninederivatives (III) and N-alkanoyl-L-carnosine derivatives (IV) to be safeand cheap carnosinase inhibitors for practical use.

Betastatin (V) or its ester derivative was also found to be a usefulcarnosinase inhibitor for the purpose of this invention as well as forcancer chemotherapy.

(R₁ and R₂ independently represent hydrogen atom or a lower alkyl group)

(R₂ represents hydrogen atom or a lower alkyl group, and R₃ representsan alkyl group of more than two carbon atoms)

(R₂ represents hydrogen atom or a lower alkyl group, and R₃ representsan alkyl group of more than two carbon atoms)

Namely, this invention relates to:

(1) combined use of a carnosinase inhibitor with L-carnosine and itsrelated substance to treat or prevent carnosine-related disorders anddiseases of humans and other mammals,

(2) combined use of a carnosinase inhibitor with L-carnosine and itsrelated substance to improve health conditions of humans and othermammals,

(3) combination of a carnosinase inhibitor with L-carnosine or itsrelated substance to improve health conditions of the skin of humans andother mammals,

(4) Combined use of a carnosinase inhibitor with L-carnosine or itsrelated substance to prevent adverse effects caused by drinking alcohol,

(5) the combined use described in (1) to (4), wherein at least one ofthe carnosinase inhibitors is selected from the group comprising ofβ-alanine (II), an N-alkanoyl-β-alanine derivative(III), anN-alkanoyl-L-carnosine derivative (IV) and bestatin (V),

(R₁ and R₂ independently represent hydrogen atom or a lower alkyl group)

(R₂ represents hydrogen atom or a lower alkyl group, and R₃ representsan alkyl group of more than two carbon atoms)

(R₂ represents hydrogen atom or a lower alkyl group)

(6) the combined use described in (1) to (4) to increase theconcentration of L-carnosine or its related substance in the plasma andthe cells of humans and other mammals to the physiologically effectivelevels,

(7) the combined use described in (1) to (4), wherein theL-carnosine-related substance is N-acetyl-L-carnosine, L-anserine,N-acetyl-L-anserine, L-balenine, L-homocarnosine,N-acetyl-L-homocarnosine or carcinine,

(8) the combined use described in (5), wherein the carnosinase inhibitoris β-alanine,

(9) the combined use described in (5), wherein N-alkanoyl-β-alanine(III) is N-acetyl-β-alanine which may be esterified by a lower alkylgroup,

(10) the combined use described in (1) to (4), wherein at least one ofthe carnosinase inhibitors is selected from the group comprising of anα-amino acid derivative, γ-amino acid derivative, a thiol type reducingagent, a metal chelating agent, a hydrophobic vitamin with a branchedhydrocarbon residue, manganese ion or zinc ion,

(11) the method of increasing the concentration of L-carnosine or itsrelated substance in the plasma and the cells to the physiologicallyeffective level, wherein at least one of the carnosinase inhibitors isselected from the group comprising of β-alanine (II), anN-alkanoyl-β-alanine derivative (III), an N-alkanoyl-L-carnosinederivative (IV), bestatin (V), an α-amino acid derivative, a γ-aminoacid derivative, a thiol type reducing agent, a metal chelating agent, ahydrophobic vitamin with a branched hydrocarbon residue, manganese ionand zinc ion,

(12) the method described in (11), wherein N-acetyl-L-carnosine,L-anserine, N-acetyl-L-anserine, L-balenine, L-homocarnosine,N-acetyl-L-homocarnosine, or carcinine is used as an L-carnosine-relatedsubstance,

(13) the method described in (11), wherein the carnosinase inhibitor isβ-alanine,

(14) a pharmaceutical composition containing L-carnosine or its relatednatural substance and at least one of the carnosinase inhibitors isselected from the group comprising of β-alanine (II), anN-alkanoyl-β-alanine derivative (III), N-alkanoyl-L-carnosine derivative(IV), bestatin (V), an α-amino acid derivative, a γ-amino acidderivative, a thiol type reducing agent, a metal chelating agent, ahydrophobic vitamin with a branched hydrocarbon residue, manganese ionand zinc ion,

(15) a health food or a dietary supplement composition containingL-carnosine or its related natural substance and at least one of thecarnosinase inhibitors is selected from the group comprising ofβ-alanine (II), an N-alkanoyl-β-alanine derivative (III), anN-alkanoyl-L-carnosine derivative(IV), bestatin (V), an α-amino acidderivative, a γ-amino acid derivative, a thiol type reducing agent, ametal chelating agent, a hydrophobic vitamin with branched hydrocarbonstructure, manganese ion and zinc ion,

(16) a health food or a dietary supplement composition described in(15), wherein the carnosinase inhibitor is β-alanine.

(17) a cosmetic or a skin-care composition containing L-carnosine or itsrelated natural substance and at least one of the carnosinase inhibitorsselected from the group comprising of β-alanine (II), anN-alkanoyl-β-alanine derivative (III), N-alkanoyl-L-carnosine derivative(IV), bestatin (V), an α-amino acid derivative, γ-amino acid derivative,a thiol type reducing agent, a metal chelating agent, a hydrophobicvitamin with branched hydrocarbon structure, manganese ion and zinc ion,

(18) alcoholic drinks containing L-carnosine or its related naturalsubstance and a carnosinase inhibitor selected from the group comprisingof β-alanine (II), an N-alkanoyl-β-alanine derivative (III),N-alkanoyl-L-carnosine derivative (IV), bestatin (V), an α-amino acidderivative, γ-amino acid derivative, a thiol type reducing agent, ametal chelating agent, a hydrophobic vitamin with branched hydrocarbonstructure, manganese ion and zinc ion,

(19) the composition described in (14) to (17), wherein at least one ofthe additives selected from the group comprising of a cellulosederivative, gelatine and polyvinyl pyrrolidone is used to increase theabsorption of L-carnosine or its related substance and/or thecarnosinase inhibitor,

(20) the composition described in (14) to (18), whereinN-acetyl-L-carnosine, L-anserine, N-acetyl-L-anserine, L-balenine,L-homocarnosine, N-acetyl-L-homocarnosine, or carcinine is used as anL-carnosine-related substance,

(21) the composition described in (14) to (18), wherein the carnosinaseinhibitor is β-alanine, and

(22) cancer chemotherapy characterized by treating cancer patients withL-carnosine or its related substance together with bestatin (V).

In the above formulae (III) and (IV), R₁ and R₂ may be the same ordifferent and each represents hydrogen atom or a lower alkyl group witha straight chain or a branched chain, preferably having 1 to 6 carbonatoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, heptyl, hexyl, etc. R₃ in the above formula (IV)represents an alkyl group having more than two carbon atoms, which maybe a straight or a branched one, such as ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl and eicosyl.

Such preferable N-alkanoyl-β-alanine derivatives (III) are exemplifiedby N-formyl-β-alanine, N-acetyl-β-alanine, N-propionyl-β-alanine,N-butyryl-β-alanine, N-(2-methylpropionyl)-β-alanine,N-pentanoyl-β-alanine, N-hexanoyl-β-alanine, N-heptanoyl-β-alanine, andtheir esters (e.g. lower alkyl esters with 1 to 6 carbon atoms such asmethyl and ethyl esters). Above all, the most preferable and convenientone is N-acetyl-β-alanine. Above mentioned N-alkanoyl-β-alaninederivatives (III) may be known compounds or can be synthesized fromβ-alanine by per se known methods such as acylation or esterification.

Such preferable N-alkanoyl-L-carnosine derivatives (IV) are exemplifiedby N-propionyl-L-carnosine, N-butyryl-L-carnosine,N-(2-methylpropionyl)-L-carnosine, N-pentanoyl-L-carnosine,N-hexanoyl-L-carnosine, N-heptanoyl-L-carnosine, and their methyl andethyl esters. Above mentioned N-alkanoyl-L-carnosine derivatives (IV)may be known compounds or can be synthesized from L-carnosine (I) by perse known methods using acylating or esterifying agents.

The bestatine derivatives represented by the above formula (V) areexemplified by bestatin and its esters such as methyl ester, ethyl esterand propyl ester.

In addition to the above carnosinase inhibitors (II, III, IV and V), thefollowing compounds may also be used for the purpose of the presentinvention: α-amino acids and their derivatives such as L-alanine,L-leucin, L-isoleucin, L-valine, L-methionine, L-phenylalanine,N-acetyl-L-cysteine, N-aminoacyl-L-histidine (e.g. Gly-L-His,L-Ala-L-His, L-Val-L-His, L-Leu-L-His, L-Ileu-L-His, L-Met-L-His, etc.),N-hydroxy-α-amino acids; γ-amino acids (e.g. γ-aminobutyrylhistamine);thiol type reducing agents (e.g. unithiol, dithiothreitol and2-mercaptoethanol); manganese ion; zinc ion; substrates of prolinase(L-proline-containing peptides such as L-Pro-His and its derivatives);metal chelating agents (e.g. EDTA); 1,10-phenanthroline; butyl malonate;and so on.

Furthermore, some hydrophobic compounds such as vitamin A and vitamin Ewith the branched hydrocarbon residue can also be used as thecarnosinase inhibitor(s).

As L-carnosine and its related substances, namely one of the activeingredients in this invention, natural substances such as L-carnosine,L-anserine, L-balenine, L-homocarnosine, carcinine and their acylderivatives (e.g. N-acetyl-L-carnosine, N-acetyl-L-anserine,N-acetyl-L-homocarnosine, etc.) are exemplified. The acyl derivativesmay be prepared by per se known method using acylating agents.

As L-carnosine is the major β-alanine source in the human body, itshydrolysis is of metabolic importance. In humans, this reaction iscatalyzed by two isozymes: tissue (cytosolic) carnosinase and serumcarnosinase. The tissue enzyme is a zinc dependent metalloprotein, and,interestingly, it can be stabilized by other divalent metal cations.Thus the replacement of Zn(II) in the enzyme by some other divalentcations maintains or increases the enzyme activity in vitro. Theapparent efficiency of activation by the divalent metal ions is given bythe order Cd(II)>Mn(II)>>Zn(II)>Co(II). On the other hand, the enzymeactivated by the Mn(II) is inhibited either by cations functioning asactivators (e.g. Zn(II), Co(II)) or by non-activating ions such asBe(II) and Fe(II). Cu(II) does not inhibit the enzyme due to thestability of the copper complex of the substrate. Thus the easiest wayto protect L-carnosine or its related natural substances from hydrolysisby tissue or serum carnosinase for nutritional purpose would be thecombination of the oral L-carnosine with Zn(II) or Mn(II) ion. Oraldaily dose of about 400 mg of L-carnosine or another natural derivativesuch as anserine and balenine admixed with about 5 mg of Zn(II) orMn(II) is generally recommended for adult human, although the doses canbe varied with the purpose of the use. Additionally, vitamin E (40-100-IU) can be added to the same oral formulation to further increase thebioavailability of L-carnosine.

A unique feature of serum carnosinase should be noted. It is presentonly in higher primates (man and the great apes) but not in most othermammals (M. C. Jackson et al., Clin. Chim. Acta, Vol. 196, 193, 1991).Accordingly, the purpose of combined application of L-carnosine or itsrelated natural substances with carnosinase inhibitor(s) in equines andcanines is to increase the bioavailability of L-carnosine or its relatedsubstances in tissues by inhibiting tissue carnosinase in these species.

The carnosinase inhibitors (II), (III), (IV), (V) or others mentionedabove may be administered either at the same time or just before orafter the administration of L-carnosine or its related substances, or asa composition combined with these compounds.

The carnosinase inhibitors and L-carnosine or its related naturalsubstances or a composition containing thereof may be administered inthe form of proper formulations such as powder, granules, capsules,tablets, injection, instillation, lozenges, solutions, ointment,cataplasm, lotion, cream, spray, nasal spray, nasal drops, eye drops,suppository, etc. depending on the purpose of the use. Theseformulations can be made according to per se known methods by addingproperly selected additives and diluents.

In order to prepare liquid formulations suitable for oral use, additivessuch as water; sugars (e.g. sucrose, sorbit, fructose); glycols (e.g.polyethylene glycol, propylene glycol); oils (e.g. sesame oil, oliveoil, soy oil); and preservatives (e.g. p-hydroxybenzoic acid esters) canbe used.

In order to prepare solid formulations (e.g. capsules, tablets, powders,granules), diluents (e.g. lactose, glucose, sucrose, mannit); collapsingagents (e.g. starch, sodium alginate); smoothers (e.g. magnesiumstearate, talc); binders (e.g. polyvinyl alcohol, hydroxypropylcellulose, gelatin); surfactants (e.g. fatty acid esters); andplasticizers (e.g. glycerin) can be used.

Among the formulations suitable for parenteral treatment, a solutionformulation for intra-venous injection or instillation, may be preparedby using an aqueous medium which is preferably osmometrically equal tothe living body. Saline, glucose solution or a mixture thereof may beused as such medium together with suitable additives to prepare asolution, suspension or dispersion by the usual methods.

Eye drops can be prepared by using an aqueous solvent such as a sterilepurified water or saline, or by using a non-aqueous solvent such asvegetable oil.

In order to prepare suppository for intestinal application, carrierssuch as cocoa butter, hydrogenated fat or hydrogenated carboxylic acidcan be used.

Spray formulations can be prepared by using carriers which do notirritate the mucous membranes in the mouth and the respiratory tracts,and make the compounds in this invention distribute well as fineparticles to accelerate their absorption from the mucous membranes. Suchcarriers are exemplified by lactose and glycerin. Depending on theproperties of the compound in this invention, aerosol or dry powderformulations may be prepared.

In order to prepare formulations for non-oral treatment, one or morethan two additives selected from diluents, essences, preservatives,excipients, disintegrants, smoothers, binders, surfactants,plasticizers, etc.

Ointment formulation may be prepared by mixing the compounds in thisinvention with suitable base such as oily base (e.g. Vaseline, liquidparaffin, silicone, vegetable oil), emulsion base (e.g. water-miscibleVaseline, purified lanolin, and water-soluble base (e.g. macrogol).Emulsifying agents (e.g. negative ionic or non-ionic surfactant) andpreservatives (e.g. p-hydroxybenzoic acid esters) may also be added inthe ointment.

Other formulations to apply the compounds of this invention to the skinas exemplified by patches, pap, creams, hard ointments, tapes, lotions,solutions, suspensions, emulsions and sprays may be prepared.

In order to enhance the absorption of L-carnosine and the carnosinaseinhibitor, it is preferable to add at least one additive selected fromcellulose derivative (e.g. hydroxypropylcellulose), gelatin andpolyvinylpyrrolidone. The form as pharmaceutical and the method of itspreparation in this invention are not limited to the above mentionedexplanation.

L-Carnosine (or its related substance) and a carnosinase inhibitor canbe added to alcohol-containing drinks such as, whisky, cognac, vodka,beer, sake, and wine to protect the drinkers from various damages of theintestinal tract, the liver, muscles and the brain.

The daily dosage of L-carnosine or its related substances varies withthe purpose of its use and the route of its administration but its oraldose is generally in the range of about 0.1 to 200 mg per kg bodyweight, preferably about 0.5 to 100 mg per kg body weight, morepreferably about 1 to 50 mg per kg body weight. The dose, if necessary,may be divided into several times a day. Its dose for injection orinstillation is usuallyabout onefifth toone twentieth of the dose of theabove oral dose. In the case of formulations for external application,about 0.1 to 20%(w/w), preferably about 0.5 to 10% (w/w), morepreferably about 1 to 5% (w/w) of L-carnosine or its related substancemay be included in the formulations. The amount of carnosinaseinhibitors to be used simultaneously is in the range of about 0.1 to 20mole equivalent, preferably about 0.5 to 10 mole equivalent, morepreferably about 1 to 5 mole equivalent per mole of L-carnosine. Thedoses of L-carnosine or its related natural substance and a carnosinaseinhibitor to be administered to mammals other than humans are similarlyin the dose range for humans as mentioned above.

According to the method of the present invention, L-carnosine or itsrelated substances administered to humans and other mammals can beeffectively delivered to and distributed in the plasma and the targettissues or organs without degradation by carnosinase.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are shown to illustrate the scope of thisinvention.

EXAMPLE 1 Hydrolysis of L-Carnosine and Related Substance with a TissueCarnosinase in the Absence or the Presence of a Carnosinase Inhibitor

Materials and Methods:

Partially purified tissue carnosinase as a solution in 0.1M phosphatebuffer (pH 7.0) is prepared from the crude brain extracts of maleHairless rats by a reported procedure (Y. Courbebaisse, G. Langrand, J-F. Nicolay and M. A. Babizhayev, Proceeding of 19th IFSCC Congress,Australia, Vol. 3, 1996, p. 1-12; M. A. Babizhayev et al., Letters inPeptide Science, Vol. 5, 163, 1998). A solution of the substrate (450μl) in 0.1M phosphate buffer (pH 7.0) is incubated with the dilutedenzyme solution (50 μl) at 37° C. for 120 min. in the absence or in thepresence of a carnosinase inhibitor. Portions of the reaction mixtureare taken at the time points of 0, 2, 3, 4, 5, 10, 20, 40, 60 min anddansylated with dansyl chloride (5 mg of dansyl chloride/ml ofacetonitrile solution) at 50° C. for 15 min. The each portion issubjected to HPLC analysis on a column C18DB using a gradient method(A=0.02N acetic acid/acetonitrile, 90/10; B=acetonitrile). The rate ofthe peptide hydrolysis is determined by following the decrease of thearea under the curve (AUC) of the dansylated substrate.

Results:

The rates of hydrolysis are as follows: L-Carnosine (0.3 mM): 78 × 10⁻⁶mole/min (blank test) L-Carnosine (0.3 mM) + 54 × 10⁻⁶ mole/minβ-alanine (0.5 mM): L-Carnosine (0.08 mM): 34 × 10⁻⁶ mole/min (blanktest) L-Carnosine (0.08 mM) + 78 × 10⁻⁶ mole/min (blank test)L-carnosine (0.3 mM): L-Carnosine (0.08 mM) + 5 × 10⁻⁶ mole/minβ-alanine (0.5 mM): L-Carnosine (0.08 mM) + 6 × 10⁻⁶ mole/min Gly-L-His(0.5 mM): L-Carnosine (0.08 mM) + 4.5 × 10⁻⁶ mole/min γ-aminobutyryl-histamine (0.5 mM): L-Carnosine (0.08 mM) + 9 × 10⁻⁶ mole/minL-Pro-L-His (0.5 mM): L-Carnosine (0.08 mM) + 3 × 10⁻⁶ mole/minN-acetyl-β-alanine (0.5 mM): N-Acetyl-L-carnosine (0.08 mM): 1 × 10⁻⁶mole/min (blank test) N-Acetyl-L-carnosine (0.08 mM) + 0.35 × 10⁻⁶mole/min N-acetyl-β-alanine (0.5 mM): L-Carnosine (0.08 mM) + 2 × 10⁻⁶mole/min bestatine (0.5 nM):

Conslusion:

As seen in the above results, it appeared that L-carnosine or therelated substance was metabolized (hydrolyzed) less quickly in thepresence of a tissue carnosinase inhibitor. This observation indicatesthat the inhibition of the enzyme with tissue carnosinase activity is atleast partially responsible for the delay of the intracellularmetabolism and for the accumulation of the natural histidine-containingdipeptides, when L-carnosine or a related substance is administeredcombined with the claimed inhibitor in vivo.

EXAMPLE 2 Combined use of L-Carnosine and β-Alanine in Horses

Five thoroughbred horses were fed 3 times/day with β-alanine (100 mg/kgbody weight) and L-carnosine (80 mg/kg body weight) for a period of 25days. In a separate study, another group of 5 thoroughbred same bredhorses were treated similarly with L-carnosine (80 mg/kg body weight)only for the same period of 25 days. Percutaneous biopsies of the m.gluteus medius from a depth of 6.2 cm were taken on the days immediatelybefore and after the supplementation period. Heparinized blood sampleswere also collected at hourly intervals on the first and last days ofsupplementation, and 2.5 h after ration on every fifth day. Individualmuscle samples were dissected from freeze-dried biopsies, weighed andcharacterized. The concentrations of β-alanine and L-carnosine in plasmaand muscles were measured by reported methods (J. J. O'Dowd et al.,Biochim. Biophys. Acta, Vol. 967, 241, 1988; M. Dunnett and R. C.Harris, J. Chromatography B, Vol. 688, 47, 1997). The areas under theplasma concentration-time curves (AUC) for β-alanine and L-carnosinewere calculated as indicators of the doses absorbed. Muscle fibrecarnosine concentrations increased from 60.5-105.5 mmol/kg (means)±(S.D.equal to 5-8% of the mean) dry weight in L-carnosine fed horses to88.9-125.5 mmol/kg (means)±(S.D. equal to 4-6% of the mean) dry weightin β-alanine+L-carnosine fed animals. The increases were statisticallysignificant for the average data in 4 of the 5 horses in each group(P<0.05). Changes in muscle L-carnosine concentration appeared to beaffected by the bioavailability of β-alanine. Individual increases inmuscle L-carnosine concentration were significantly correlated withindividual changes in areas under the plasma-concentration time curves(r=0.895, P<0.05) of β-alanine.

Discussion:

It was reported that the increased muscle L-carnosine concentrationscorrelated with the increased muscular daily activity of horses adaptedto either high-speed running or to prolonged periods of hypoxia (M.Dunnett and R. C. Harris, J. Chromatography B, Vol. 688, 47, 1997).Under these conditions, the muscle contraction is known to be reliantupon the anaerobic glycolysis for rapid ATP turnover and to beassociated with a large increase in muscle lactic acid concentration.The accumulation of H⁺ from lactic acid dissociation within skeletalmuscle would produce a critical fall in intra-cellular pH in the absenceof endogenous H⁺ buffering substances. Progressive intracellularacidosis may be a primary cause of local muscle fatigue. The imidazoledipeptides (pK_(a) 6.8-7.1) function as important H⁺ buffers over thephysiological pH range (R. C. Harris et al., Comp. Biochem. Physiol.,Vol. 97A, 249, 1990). Higher L-carnosine concentrations in muscle fibresare consistent with their higher capacity for H⁺ production and thusgreater buffering requirement (M. Dunnett and R. C. Harris, J.Chromatography B, Vol. 688: 47, 1997). The combined oral administrationof L-carnosine with β-alanine, an inhibitor of carnosinase, is asuitable way to increase the concentration of the imidazole-containingdipeptide, L-carnosine, in the muscular tissues.

EXAMPLE 3 Induction of Apoptosis in the Cancer Cells Upon Application ofL-Carnosine with a Carnosinase Inhibitor

The experimental design, details of experimental procedure and humanleukemia cell lines were essentially the same as those reported bySekine et al. (Int. J. Cancer, Vol. 94, 485, 2001). The carnosinaseinhibitor bestatine was used in the range of 5-15 μg/ml of concentrationand the concentration of the applied carnosine varied from 5-20 mM . Thecombined use of L-carnosine (10 mM) and the tissue carnosinase inhibitorbestatin (10 μg/ml) induced apoptosis in several human leukemia celllines. Our study was performed with this combination to examine whetherbestatin+carnosine can also induce apoptosis in solid tumor cell lines.Bestatin alone at above-cited concentrations exhibited neither directgrowth inhibition nor induction of apoptosis in the tumor cell linesexamined. However, the composition of L-carnosine+bestatin significantlyaugmented the growth-inhibitory effect and induction of apoptosis to thecited tumor cell lines. When used with L-carnosine, bestatin methylester, a more cell-permeable bestatin derivative with similar inhibitoryactivity against cytosolic tissue carnosinase, inhibited cell growthmore clearly than the case where only L-carnosine is used. In ourstudies according to the above-cited design, the combination of 10 μg/mlof bestatin with 10 mM L-carnosine promoted processing of caspase 3 tothe active form p17 and efflux of mitochondrial cytochrome C into thecytosol in solid tumor cells and in several human leukemia cell lines.

Conslusion:

These results suggest that intracellular administration ofcarnosine+carnosinase inhibitor might play an important role in theapoptosis of the solid tumor and other cell lines

EXAMPLE 4 Addition of L-Carnosine and a Carnosinase Inhibitor to theAlcoholic Drink

We have used the newly composed Extra Vodka Corn including L-carnosineand β-alanine which was manufactured at the Russian Plant Corn & Co. bymaking a solution of 70 mg/200 g of each ingredient in the vodka withvigorous stirring. The taste of and the euphoria properties of the vodkaunchanged, and no side reactions like headache or liver toxicity wereobserved upon 20 days of the assumption by the Managers of this Plantwho taste the vodka drink regularly. Five volunteers used the composedvodka drink each day at a dose of 200 g. The level of acetaldehyde andmalonaldehyde were not increased in the urine at the end of the trial onthe 20^(th) day in the morning one hour after drink as compared to thosemeasured prior to the study.

EXAMPLE 5 Oral Care Application of L-Carnosine with a CarnosinaseInhibitor

L-Carnosine and β-alanine were applied to 7 men subjects with gingivitisas a combined, oral formulation. The composition of carnosine+β-alaninewas found to stimulate the healing of gingival tissues and completelyabolished bleeding. The reaction of inflammation was decreased. Thecited composition of carnosine 5 mg/ml+β-alanine (7 mg/ml) demonstratedsignificantly increased granulation in the tooth gingival tissues. Thedata on the use of L-carnosine only for the wound healing of gingivaltissues have been published earlier (K. Nagai, Langenbecks Arch. Chir.,Vol. 351, 39, 1980; T. Yamane et al., J. Nihon Univ. School. Dent., Vol.19, 70, 1977).

EXAMPLE 6 Application of L-Carnosine with the Carnosinase Inhibitorβ-Alanine in Cosmetic Composition as a Pigment Tanning Product

This composition can induce the pigment adaptation in dark brown, blondand pigmented hair and in the skin. The cosmetic composition ofβ-alanine with carnosine was heated with phospholipid lecithin toproduce the sun tanning pigment in the skin. β-Alanine does not producethe coloring in the solution per se and can produce the coloring by thereactions with tyrosine and tyrosinase-oxidized tyrosine, dopa ordopamine.

Preparation of the cosmetic formulation (described in: Babizhayev M. A.Antioxidant activity of L-carnosine, a natural histidine-containingdipeptide in crystalline lens. Biochim. Biophys. Acta 1004 (1989)363-371):

A mixture of 1% carnosine and 2% β-alanine residues were included in theliposomes of lecithin (derived from egg yolk phosphatidylcholine)prepared by reverse phase evaporation. The liposomes were formed from anemulsion of phospholipid in the phosphate buffered solution loaded withthe cited compounds with a modified procedure including the two stepssimultaneously, i.e., mixing of heptane and buffer solution and removalof organic solvent. In a standard preparation 100 mg ofphosphatidylcholine was dissolved in 15 ml heptane in a round-bottomedflask and 5 ml of 0.1 mol/l (or less concentrated) PBS buffer (pH 7.4)was added. The flask was immersed in a water-bath warmed to 50° C. Afterthorough mixing nitrogen was poured through a glass capillary into thesample. Heptane was usually removed at a rate of 0.5-1.0 ml·min⁻¹. Weobserved an enhancement of the viscosity of the mixture up to apaste-like state. After that, the gel collapsed relatively fast and aturbid nonviscous liposome suspension was formed. Single gel dropletsdisappeared after further nitrogen pouring. The mean hydrodynamicdiameter of vesicles in the prepared cosmetic composition was 350 nmwith a polydispersity of 27%.

The composition was daily applied on the skin of human elderlyvolunteers (10 subjects) for 15 days twice daily and showed pigmentaryadaptation to produce a smooth tan pigment including the disappearanceof age-related lipofuscin-like spots on the skin. The spots were presentoriginally in the volunteers or were then were substituted with thetanning pigment upon the treatment. The fluorescence of the skin in theblue green zone (measured with spectrofluorophotometer equipped with adistant opto-fiber probe) was decreased and good/fresh appearance of theskin increased. Both spectra of excitation and emission of fluorescenceat 365 nm excitation wavelength were monitored and the intensity offluorescence of lipofuscin-associated pigments was decreased. Thesubjects underwent with the cited treatment reported the decrease of thespot lipofuscin-like age pigments previously exposed on the skin. Thepure carnosine application showed only a little whitening activityduring the experiment. The observed disappearance of lipofuscin-relatedspots on the skin is related to the action of carnosine as the de-linkerof cross-links in the skin proteins combined with the tanning activityof β-alanine.

EXAMPLE 7 Treatment of Autism

For autistic children, most beneficial effects were obtained whentreated orally with a dosage of 400 mg L-anserine in combination with50-IU Vitamin E and 5 mg zinc twice a day. In some children, too highdose may overstimulate some patient's frontal lobes which may causeincreased irritability, hyperactivity or insomnia which was observedalready in hyperactive autistic children.

EXAMPLE 8

Treatment of Neural and Brain Disorders

Children with neural disorders such as epilepsy, central processingdisorder, or brain injury were able to be treated with oral daily doseof 200 to 2000 mg of L-carnosine in combination with 40-IU to 150-IUVitamin E and 4 to 15 mg zinc.

EXAMPLE 9

The following formulations are made by per se ordinary procedures:

1. Cosmetic formulation:

A hydroglycolic solution of which analytical composition is: Carnosine10.000 g β-alanine 15.000 g 1,3-Butanediol 8.180 g Sodium methyl paraben0.145 g Water up to 100.000 g

The composition is a limpid liquid, colorless, with a light butanediolodor which is miscible with water, glycols and alcohol, but not misciblewith hexane, mineral and vegetable oils.

2. Oral formulation for dietary supplement:

Capsulated formulation in one gelatine or rice capsule: L-Carnosine  80mg β-alanine 100 mg

3. Ophthalmic formulation for oxidative stress-induced ocular disorders:L-Carnosine 1.00 g β-alanine 1.00 g Dibasic sodium phosphate 0.80 gMonobasic sodium phosphate 0.15 g Benzalconium chloride 0.010 g Purifiedwater up to 100 ml

4. Alcoholic drink: 40% Vodka 500 ml L-Carnosine 400 mg β-alanine 600 mgWater up to 1000 ml

INDUSTRIAL APPLICABILITY

The present invention aims to provide combined use of a carnosinaseinhibitor with L-carnosine and its related substance and a compositioncontaining the same, which are useful for treatment or prevention ofdiseases and improvement of health conditions. The present inventionalso provides a method for prevention or treatment of carnosine-relateddiseases, a method for improvement of health conditions, a method forimprovement of exercise performance, a method for improvement of skinhealth and a method for prevention of the side effects of alcoholdrinking, comprising combined use of a carnosinase inhibitor withL-carnosine and its related substance, for mammals. The presentinvention is useful in the fields of pharmaceuticals, cosmetics, healthfoods, foods and beverages.

1-22. (canceled)
 23. A composition comprising L-carnosine or its relatedsubstance and at least one carnosinase inhibitor.
 24. The compositionaccording to claim 23, wherein the carnosinase inhibitor is selectedfrom the group comprising of β-alanine (II), an N-alkanoyl-β-alaninederivative (III), an N-alkanoyl-L-camosine derivative (IV), bestatin (V)or a derivative thereof, an α-amino acid or a derivative thereof, aγ-amino acid or a derivative thereof, a thiol type reducing agent, ametal chelating agent, a hydrophobic vitamin with a branched hydrocarbonresidue, manganese ion and zinc ion.
 25. The composition according toclaim 23, wherein the composition is a pharmaceutical composition. 26.The composition according to claim 23, wherein the composition is ahealth food or a dietary supplement.
 27. The composition according toclaim 23, wherein the camosinase inhibitor is β-alanine.
 28. Thecomposition according to claim 23, wherein the composition is a cosmeticor a skin-care composition.
 29. The composition according to claim 23,wherein the composition is an alcoholic drink.
 30. The compositionaccording to claim 23, wherein the composition further includes at leastone additive selected from the group comprising of a cellulosederivative, gelatin and polyvinyl pyrrolidone, wherein the additiveincreases the absorption of L-carnosine or its related substance and/orthe carnosinase inhibitor.
 31. The composition according to claim 23,wherein the L-carnosine related substance is N-acetyl-L-carnosine,L-anserine, N-acetyl-L-anserine, L-balenine, L-homocarnosine,N-acetyl-L-homocamosine, or carcinine is used as an L-carnosine-relatedsubstance.
 32. the composition according to claim 23, wherein thecarnosinase inhibitor is bestatin (V).
 33. The composition according toclaim 23, wherein the β-alanine (II), the N-alkanoyl-β-alaninederivative (III), the N-alkanoyl-L-carnosine derivative (IV) andbestatin (V) have the following formulas:

wherein R₁ and R₂ independently represent hydrogen atom or a lower alkylgroup;

wherein R₂ represents hydrogen atom or a lower alkyl group, and R₃represents an alkyl group of more than two carbon atoms; and

wherein R₂ represents hydrogen atom or a lower alkyl group.
 34. Thecomposition according to claim 33, wherein the N-alkanoyl-β-alanine(III) is N-acetyl-β-alanine which may be esterified by a lower alkylgroup.
 35. A method of increasing the concentration of L-carnosine orits related natural substance in the plasma or cells of a human or othermammal, which comprises administering to the human or other mammal aneffective amount of the composition according to claim
 23. 36. A methodof treating or preventing camosine-related disorders or diseases in ahuman or other mammal, which comprises administering to the human orother mammal an effective amount of the composition according to claim23.
 37. A method for improving a health condition of a human or othermammal, which comprises administering to the human or other mammal aneffective amount of the composition according to claim
 23. 38. A methodfor improving a skin condition of a human or other mammal, whichcomprises administering to the human or other mammal an effective amountof the composition according to claim
 23. 39. A method for preventing anadverse effect caused by drinking alcohol, which comprises administeringto the human or other mammal an effective amount of the compositionaccording to claim
 23. 40. A method for treating cancer in a human orother mammal, which comprises administering to the human or other mammalan effective amount of the composition according to claim 23, whereinthe camosinase inhibitor is bestatin (V).